The broad objectives of this project are: (1) to understand better the molecular basis of state-dependent interactions between voltage-gated Na channels and local anesthetics (LAs) and (2) to explore the interplay between LAs and the Na+ permeation pathway. This pathway, in part, consists of a selectivity filter, permenant ion binding sites and as inactivation gate. Among LAs included are putative inactivation enhancers, such as benzocaine and tricaine, putative open-channel blockers, such as cocaine, bupivacaine, and quaternary ammonium (QA) compounds, and putative dual blockers such as tetracaine and procaine. In this proposal, we plan to examine the structural basis that distinguishes these three distinct LA types. Two separate hypotheses will be tested: first, only one single receptor is present within the Na+ permeation pathway for all three types of LAs and second, the common amino group on the phenyl ring of inactivation enhancers and dual blockers preferentially stabilizes the inactivated state of the Na+ channel. Both whole-cell and single channel currents will be measured in order to obtain detailed kinetic information on the dynamic interactions between Na+ channels and LAs. Because ion-ion repulsion within the pore is a common trait for ion permeation, demonstration and further characterization of a knock-out phenomenon of LA/QA ions by the inflowing cations through the Na+ selectivity filter will be obtained to provide crucial evidence that the LA binding site is indeed located within the Na+ permeation pathway. Concurrently, we will delineate LA- channel interactions at the molecular level. At first, the LA binding toward cloned mu1 muscle Na+ channels will be studied with and without Beta1 subunit present. Subsequently, the roles of two separate regions of mu1 Na+ channels, including the internal QA binding site (probably within the pore and S6 regions) and the inactivation-related loop (between domain III and IV), on LA binding affinities will be examined by the macropatch technique in Xenopus oocytes injected with wild-type and mu1 mutant mRNAs. Together, these studies should provide a clearer understanding of LA-Na+ channel interactions as well as the whereabouts of the LA/QA binding site within the Na+ permeation pathway.